TY - JOUR
AU1 - Hopkins, Brandon, K
AU2 - Chakrabarti,, Priyadarshini
AU3 - Lucas, Hannah, M
AU4 - Sagili, Ramesh, R
AU5 - Sheppard, Walter, S
AB - Abstract Global decline in insect pollinators, especially bees, have resulted in extensive research into understanding the various causative factors and formulating mitigative strategies. For commercial beekeepers in the United States, overwintering honey bee colony losses are significant, requiring tactics to overwinter bees in conditions designed to minimize such losses. This is especially important as overwintered honey bees are responsible for colony expansion each spring, and overwintered bees must survive in sufficient numbers to nurse the spring brood and forage until the new ‘replacement’ workers become fully functional. In this study, we examined the physiology of overwintered (diutinus) bees following various overwintering storage conditions. Important physiological markers, i.e., head proteins and abdominal lipid contents were higher in honey bees that overwintered in controlled indoor storage facilities, compared with bees held outdoors through the winter months. Our findings provide new insights into the physiology of honey bees overwintered in indoor and outdoor environments and have implications for improved beekeeping management. honey bee overwintering, indoor storage, honey bee physiology, head protein content, abdominal lipid content Global declines in honey bee (Apis mellifera L.) and native bee populations have been reported for more than a decade (Frazier et al. 2008, Cameron et al. 2011, Kulhanek et al. 2017). Both beekeepers and growers, interdependent on pollination services, are affected by such unsustainable colony losses. Most of the colony losses occur during the winter months (vanEngelsdorp et al. 2012), when there is little to no natural forage, and when (in colder regions) colonies survive on honey stores and reduce or halt brood rearing. The demand for strong colonies in February, to meet the requirements for almond pollination, results in the movement of more than 2 million managed colonies from across the United States to California in the winter months (NASS 2017). California almond production was predicted to need as many as 2.67 million honey bee colonies in 2019 (Ullmann et al. 2017, CDFA 2018). Even with an estimated increase of 3% in the number of honey bee hives in the United States to 2.89 million colonies (NASS 2017), the demand of 2.67 million colonies and a supply of 2.89 million shows that almonds will require more than 90% of all the managed colonies in the United States to adequately pollinate the crop in coming years. Many of these colonies are placed in ‘holding yards’ (Stokstad 2007). These holding yards have a high colony density, where beekeepers stimulate brood production by feeding sugar syrup and supplementing protein diets (Sagili and Breece 2012) to maintain or increase colony strength to meet pollination needs. However, beekeepers have often reported their greatest losses during winter months in these holding yards (vanEngelsdorp et al. 2011). The cohort of worker bees that are produced during late fall, physiologically transform to winter bees and store nutrients in their bodies, are referred to as ‘diutinus’ bees (Münch and Amdam 2010). The altered behavior and physiology of these individual honey bees help shape the overwintering state of the colony. Individually, these honey bees may show signs of reduced activity, increased nutrient stores in the body tissues, and increased longevity (Döke et al. 2015). However, changes at the colony level include complete absence of brood rearing and formation of thermoregulatory clusters (Döke et al. 2015). There is evidence that Varroa mite levels, virus titers, geographic locations, and hive genetics play an important role in winter colony losses (Genersch et al. 2010, Dainat et al. 2012, Nazzi et al. 2012, van Dooremalen et al. 2012, Pirk et al. 2014), but the role of nutritional physiology of overwintering bees under various winter storage conditions is still largely unknown. The importance that lipid and protein stores in honey bee tissues play in survival during the winter months cannot be underestimated. It has been reported that upon emergence, the longer-lived winter diutinus bees have greater weights and higher protein contents, compared with the shorter-lived worker honey bees reared during other times of the year (Kunert and Crailsheim 1988). Even though a previous study has shown that the vitellogenin protein levels in honey bees are high during the onset of winter (Fluri et al. 1982), levels of vitellogenin and hemolymph proteins are overall larger in nurse bees and overwintering bees than in the foragers (Fluri et al. 1977, 1982; Fluri and Bogdanov 1987; Döke et al. 2015). Head protein content may be a good indication of hypopharyngeal gland functioning in overwintering bees, as hypopharyngeal glands are located in the head (Buttstedt et al. 2014). Vitellogenin is synthesized by fat body cells (Bellés 2003, Corona et al. 2007), is positively correlated to longevity (Seehuus et al. 2006, Amdam et al. 2012), and is one of the most important drivers of colony strength. Vitellogenin is also a good predictor of colony winter survival (Dainat et al. 2012). The amount of lipid stores in honey bees may be a good indication of the vitellogenin titers in overwintering diutinus bees, as lipid stores increase in the diutinus phase and vitellogenin is produced by the fat body (Engels 1974, Chapman 1998, Smedal et al. 2009). Following prolonged forage scarcity through winter, the nutrients stored in winter bees are utilized to provide brood food to larvae in spring (Ricigliano et al. 2018). Hence, understanding the nutritional physiology of overwintered bees is vital in evaluating the potential impacts on colony strength and survival during ensuing spring. With commercial colony losses averaging 30–40% in the United States (Kulhanek et al. 2017), many commercial beekeepers are looking for alternatives to overwinter bees successfully. One of these includes the storage of colonies in temperature-controlled buildings, such as industrial potato sheds, purpose-built bee storage buildings, or tree-fruit storage facilities. The methods of maintaining temperatures vary from evaporative cooling to refrigeration. The primary purpose of such holding facilities is to ensure ideal storage of thousands of colonies at a steady temperature (~4°C) from late fall or early winter, until they are needed for almond pollination (Hopkins 2020). The storage of bees in buildings with evaporative cooling (e.g., potato sheds) has been the most common practice, but new buildings are generally equipped with refrigeration capacity. The use of controlled atmosphere facilities for storing bees is rare. Although the practice of storing bees indoors is not new (Doolittle 1902), and has been utilized regularly in Canada and other countries for decades (Nelson and Henn 1977, Fingler and Small 1982, McCutcheon 1984, Murrell and McDonald 1986), there are no published studies reporting the effects of wintering strategies on honey bee nutritional physiology profile. In this study, we examined the nutritional physiology (abdominal lipids and head proteins) of a marked age cohort of honey bees, before and after the winter, in various overwintering scenarios to understand the impacts of different overwintering strategies on overwintering bees. Materials and Methods Experimental Design Frames of capped brood were removed in late October 2015, from full-sized honey bee (Apis mellifera L.) colonies housed at the Washington State University apiaries (Pullman, WA) and placed in screened cages in the incubator (Darwin Chambers, KB034, St. Louis, MO) maintained at controlled conditions (30°C, 55% RH). Each day over the course of 5 d, newly emerged bees were removed from the frame cages and thoraces were painted with enamel paint (Testers, Illinois). All painted bees were held and transported to the field sites in a four-frame queen right nucleus hive at the end of the 5-d emergence and painting period. Twenty-four equalized foster colonies were palletized in a commercial beekeeping operation for this experiment, with six colonies placed on each pallet. Equalization was performed by the commercial beekeeper collaborator, so that all 24 colonies were queen right, contained 16 frames of bees, a minimum of four frames of brood and had enough weight to make it through winter. Each foster colony received 240 ml of painted bees (approximately 600 bees; Lee et al. 2010). At the same time point, three sets of 20 bees each were frozen in −80°C freezer (Thermo Fisher) to serve as time zero control group. On 24 November, each of the four pallets with honey bee colonies was sent to a different location for the winter storage period. One pallet was sent to a holding yard near Oakdale, CA. The second pallet was placed outside in the open in Naches, WA, exposed to the winter conditions. The third pallet was held in a cold storage (refrigerated) facility (4°C, Naches, WA) with high levels of ventilation to maintain indoor metabolic gases at normal atmospheric levels, and the fourth pallet was placed in a tree-fruit storage facility with controlled atmosphere (Naches, WA), having limited airflow (4°C; CO2 builds to 2% each day and vented during the night; referred to hereafter as controlled atmosphere). All pallets were treated identically prior to the onset of winter treatment with 6 liter of Pro-Sweet77 (Mann Lake, Hackensack, MN) and 1 kg of Bee-Pro patties (Mann Lake, Hackensack, MN). After 62 d, when the commercial beekeeper started moving colonies to CA, all pallets were consolidated in the holding yard location near Oakdale, CA. All foster colonies survived were inspected, and a maximum of 20 painted bees were collected from each individual colony in 50-ml centrifuge tubes (Falcon, Corning Life Sciences, Tewksbury, MA) and immediately frozen on dry ice. At the time of recapture, painted bees were 97 (±2) d old. Even though a maximum of 20 painted bees were targeted for collection from each foster colony, it was not always possible to collect 20 painted bees from all the colonies due to the differential survival of painted honey bees at the end of 62 d across all 24 foster colonies. We were able to recapture painted bees from all colonies and the minimum number of bees collected from any single colony was six. Abdominal Lipid Analysis The abdominal lipid analyses were performed using a modified protocol based on Bligh and Dyer (1959). Whole abdomens of painted bees were collected and pooled for each colony, and processed for lipid analysis at the Washington State University in Pullman, WA. The number of abdomens were counted for each hive and placed in 25-ml glass beakers. Next, they were placed in a drying oven (Napco 5831) at 70°C for 48 h. The dry weight of the abdomens was then recorded and 10-ml chloroform was poured into the beakers to fully submerge the abdomens. After 24 h, the chloroform was carefully decanted and replaced with 10-ml fresh chloroform. This washing procedure was repeated three times for each sample. After three consecutive washes, the abdomens were dried for 48 h at 70°C in the oven and reweighed. The difference in dry weight before and after the three chloroform washes was indicative of the abdominal lipid weight. Analysis of Total Head Protein Contents The heads of painted honey bees were separated, pooled together for each colony, and sent to the Oregon State University (Corvallis, OR) Honey Bee Lab for head protein analysis in May 2016. Each head was analyzed individually. Ten heads of painted bees per foster colony were analyzed, when the sample contained more than 10 heads. When the sample contained less than 10 heads, all heads in the sample were used for protein analysis. Total protein content of honey bee heads was quantified using a standard bicinchoninic acid (BCA) assay (Pierce Biotech BCA Assay Kit, Thermo Scientific, IL) based on previous studies (Chakrabarti et al. 2020). Briefly, each individual head was homogenized in 100 µl of chilled phosphate-buffered saline (PBS; 10 mM phosphate, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4, Sigma–Aldrich), with one 3-mm tungsten carbide bead (Qiagen) in a Tissue Lyser II (Qiagen; two rounds of 45 s at 30 oscillations/s). Samples were then centrifuged at 18,700 g for 4 min at 4°C to pellet the debris. Next, a 1/20th dilution of the resulting supernatant in chilled PBS was made. The kit instruction manual was followed for the microplate assay protocol and the absorbance was measured on a BioTek Synergy 2 plate reader (BioTek Instruments) at 562 nm at 25°C. Total protein content (mg per bee) was then calculated using the following formula: Head protein per bee(mg)=[protein]×sample volume×dilution factor where [protein] is protein concentration (mg/ml) reported by the assay; sample volume is the total volume of the homogenized sample (here, 0.1 ml); dilution factor is amount by which the original sample was diluted (here, 20). Statistical Analysis Data were checked for normality using Shapiro–Wilk test. Comparisons between means were performed by one-way analysis of variance (ANOVA) tests followed by Tukey’s post hoc tests. All statistical tests are done in R version 3.3.0. Means are presented as ± SEM. Results Overwintering Temperatures Across Treatments Colonies moved to California experienced a mean temperature of 8.9°C in December 2015 and 10.6°C in January 2016, with average maximum of 13.9 and 14.4°C, respectively. Colonies outside in Washington State experienced a mean temperature of −1.7°C in December and −0.6°C in January, with average maximum of 1.7 and 2.2°C, respectively. The indoor pallets (held in a cold storage and in a controlled atmosphere) were both held at 4°C. Recapture of Painted Bees From Foster Colonies Approximately 600 painted bees were installed in each of the colonies for this experiment to maximize the chances of recapturing our target of 20 bees per colony. The success rate for recapture varied between treatments. The average number of bees recaptured from colonies placed outdoors in Washington and holding yard in California, cold storage and controlled atmosphere, was as follows: 6 ± 1.03, 13.6 ± 2.94, 18 ± 2, and 19.5 ± 0.5, respectively. Abdominal Lipid Contents A significant difference in the abdominal lipid content was found between the overwintered experimental groups (one-way ANOVA with Tukey’s post hoc tests, F(4,26) = 10.18, P < 0.001; Fig. 1; Supp Table 1 [online only]). Honey bees that overwintered in colonies stored in the controlled atmosphere storage facility had the highest abdominal lipid contents (3.64 ± 0.35 μg/bee) when compared with honey bees collected at time zero (2.56 ± 0.49 μg/bee), bees that overwintered in holding yard in California (1.19 ± 0.17 μg/bee), bees that were overwintered in holding yard in Naches, WA (3.22 ± 0.37 μg/bee), and bees that overwintered in cold storage (1.70 ± 0.09 μg/bee). No significant difference was observed in the abdominal lipid content of bees collected at time zero and bees that overwintered in the tree fruit controlled atmospheric storage facility (Fig. 1). Fig. 1. Open in new tabDownload slide Mean abdominal lipid contents (μg/bee) in honey bees across various overwintering conditions. Different letters denote a significant difference between treatments (P ≤ 0.05) as evident from one-way ANOVA followed by Tukey’s post hoc comparison tests. For time point zero and indoor (cold storage and controlled atmosphere) experimental groups, 20 honey bee abdomens (n = 20) were pooled in each replicate, except one replicate in cold storage (n = 6) and one replicate in controlled atmosphere (n = 17). For the two outdoor groups, 1) in California holding yards: n = 4 for three replicates, n = 6 in one replicate, and n = 8 and n = 10 in one replicate each; 2) for outdoor groups in Washington: one replicate each had n = 4, n = 4, n = 8, and n = 10, whereas three replicates had n = 20. Fig. 1. Open in new tabDownload slide Mean abdominal lipid contents (μg/bee) in honey bees across various overwintering conditions. Different letters denote a significant difference between treatments (P ≤ 0.05) as evident from one-way ANOVA followed by Tukey’s post hoc comparison tests. For time point zero and indoor (cold storage and controlled atmosphere) experimental groups, 20 honey bee abdomens (n = 20) were pooled in each replicate, except one replicate in cold storage (n = 6) and one replicate in controlled atmosphere (n = 17). For the two outdoor groups, 1) in California holding yards: n = 4 for three replicates, n = 6 in one replicate, and n = 8 and n = 10 in one replicate each; 2) for outdoor groups in Washington: one replicate each had n = 4, n = 4, n = 8, and n = 10, whereas three replicates had n = 20. Head Protein Contents There was a significant difference in the head protein contents across the experimental groups (one-way ANOVA with Tukey’s post hoc tests, F(4,27) = 5.526, P < 0.05; Fig. 2; Supp Table 2 [online only]). The head protein content of honey bees, which overwintered in the controlled atmospheric storage facility (0.705 ± 0.03 mg/bee), was significantly higher than bees from colonies that wintered outdoors in holding yards in Naches, WA (0.550 ± 0.03 mg/bee) and in California (0.554 ± 0.03 mg/bee). The head protein content was also significantly different between honey bees from time zero (0.564 ± 0.02 mg/bee) and the bees that overwintered in controlled atmospheric storage, but there was no significant difference in head protein content between honey bees from time zero and the honey bees in cold storage (0.645 ± 0.03 mg/bee). Furthermore, there was no significant difference in the head protein contents of honey bees that overwintered in the controlled atmospheric storage and the honey bees that overwintered in cold storage. Fig. 2. Open in new tabDownload slide Mean head protein contents (mg/bee) in honey bees across various overwintering conditions. Different letters denote a significant difference between treatments (P ≤ 0.05) as evident from one-way ANOVA followed by Tukey’s post hoc comparison tests. For time point zero and indoor (cold storage and controlled atmosphere) experimental groups, 10 honey bee heads (n = 10) were pooled in each replicate, except one replicate in cold storage (n = 6). For the two outdoor groups, 1) in California holding yards: n = 4 for three replicates, n = 6 in one replicate, and n = 8 in one replicate; 2) outdoor in Washington: one replicate each had n = 4 and n = 8. Fig. 2. Open in new tabDownload slide Mean head protein contents (mg/bee) in honey bees across various overwintering conditions. Different letters denote a significant difference between treatments (P ≤ 0.05) as evident from one-way ANOVA followed by Tukey’s post hoc comparison tests. For time point zero and indoor (cold storage and controlled atmosphere) experimental groups, 10 honey bee heads (n = 10) were pooled in each replicate, except one replicate in cold storage (n = 6). For the two outdoor groups, 1) in California holding yards: n = 4 for three replicates, n = 6 in one replicate, and n = 8 in one replicate; 2) outdoor in Washington: one replicate each had n = 4 and n = 8. Discussion The growing interest in wintering commercial honey bee colonies indoors as an alternative to outdoor holding yards, during the freezing winter months, has been spurred by the financial benefit of those employing this practice (Degrandi-Hoffman et al. 2019). Until now, overwintering management practice has been largely driven by communication of shared experiences among beekeepers, with limited research data available to help refine methods. This study elucidates differences in nutritional physiology among bees which overwintered in different environmental conditions. Honey bees from only the controlled atmospheric storage facility showed significant differences in both physiological parameters assessed, when compared with the honey bees from the outdoor storage conditions and the time point zero groups. We found greater amounts of head protein in bees from the indoor storage treatments (cold storage and controlled atmospheric storage) compared with the bees from the outdoor treatments (holding yards in California and Washington). Even though the difference was not statistically significant between the indoor and outdoor storage bee cohorts, except for the controlled atmospheric storage facility, this finding potentially alludes to the need to study larger sample sizes and more colonies to address this important biological significance. The hypopharyngeal and mandibular glands are located in the head and are responsible for brood food production (Kucharski et al. 1998). Diutinus bees (winter honey bees) are reported to have larger protein stores (Fluri et al. 1977, 1982, Shehata et al. 1981, Smedal et al. 2009) to provision colonies during brood rearing and overwintering (diutinus bees must provide food to the brood reared in spring). Thus, cessation in brood rearing during winter and a controlled environment (consistent low temperatures) may have contributed to the improved retention of head protein contents in overwintering bees stored indoors when compared to bees stored outdoors in holding yards. The recapture rate of painted bees in this study provided interesting findings and insight into potential changes in colony age structure during the winter months when colonies are stored in different environments. Unfortunately, we were only able to recapture a low percentage of marked bees from colonies wintered in California. However, this was not due to low colony survival, but more likely due to the fact that many of the marked bees may have started foraging after the experimental foster colonies were transported to California and held in holding yards in December and January. There are major implications for this in terms of the need for colonies to replace bees as they mature into foraging stages and are relatively short lived from that point. The abdominal lipid content was the lowest in bees that were overwintered outdoors in California holding yards, with lipid content values found to be intermediate between the time point zero group and the honey bee group stored in cold room facility. This may be due to relatively higher temperatures prevailing in California when compared to indoor storage and outdoor storage in Washington, resulting in higher bee activity and consequently lower lipid levels. The abdominal lipid content in bees that were stored in cold facility was lower than the ones stored in controlled atmospheric storage even though the temperatures (4°C) were the same. This unexpected finding needs further investigation. We speculate that this phenomenon may be either due to physiological effects of carbon dioxide that was variable in these two storage conditions or it could be due to lower Varroa infestation because of higher carbon dioxide levels in colonies stored in controlled atmospheric storage. It has been shown that mite mortality is higher in colonies that are stored indoors with restricted ventilation (higher carbon dioxide levels; Bahreini and Currie 2015). Another possible explanation may be that extreme cold temperatures (e.g., outdoor storage in Naches, WA) can force honey bees to form tight clusters, thus generating higher carbon dioxide, which may potentially explain the higher abdominal lipid contents in these honey bees. Our study thus demonstrates the need to conduct future research with larger samples sizes to further explore these observations. Head protein is an indicator of the state of the brood food producing glands in the honey bees (Kucharski et al. 1998). In life cycle of insects, fat bodies play a major role and are involved in several metabolic functions, such as related to energy reserves (Arrese and Soulages 2010), lipid storage (Olofsson et al. 2009), and synthesis of vitellogenin (Amdam et al. 2003). As higher head protein contents and abdominal lipid contents are important markers of honey bee nutritional physiology (Chakrabarti et al. 2020), their increased levels in winter bees from the controlled indoor storage facilities suggests enhanced bee fitness and performance during the next spring. There is an increasing interest in storing bees indoors during winter months, evidenced by the reports of increased colony survival by those adopting this practice (Degrandi-Hoffman et al. 2019). Although there are a number of reasons for commercial beekeepers to adopt this strategy, there exists a gap in understanding the impacts of such overwintering storage practices on honey bee physiology. Even though a direct measure of various molecular parameters for the diutinus phase, e.g., vitellogenin titers and juvenile hormones, is beyond the scope of the present study, this leaves an opportunity for future research to examine these parameters in marked worker honey bees through the diutinus phase. Overall, our study results provide new insights into the physiology of honey bees that overwintered in both indoor and outdoor environments. The information gleaned from this study could be used to make improvements in current techniques of wintering bees indoors in controlled environment. Further research is needed to understand the effects of various management practices (e.g., appropriate time to move bees into indoor facility) on other physiological aspects of diutinus bees in the environmentally controlled indoor storage facilities. Supplementary Data Supplementary data are available at Journal of Economic Entomology online. Supplementary Table 1. Results from ANOVA Tukey’s post hoc tests for abdominal lipid content analysis. Supplementary Table 2. Results from ANOVA Tukey’s post hoc tests for head protein content analysis. Acknowledgments We acknowledge beekeepers, Matthew Shakespeare and Eric Olson, for access to honey bee colonies and management, transportation, and storage of colonies used in this research. Funding was provided by the United States Department of Agriculture National Institute of Food and Agriculture Pollinator Health: Research and Application (grant 2019-67013-29348/project accession no. 1018969). 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TI - Impacts of Different Winter Storage Conditions on the Physiology of Diutinus Honey Bees (Hymenoptera: Apidae)
JF - Journal of Economic Entomology
DO - 10.1093/jee/toaa302
DA - 2021-02-09
UR - https://www.deepdyve.com/lp/oxford-university-press/impacts-of-different-winter-storage-conditions-on-the-physiology-of-SOeIQkCLFA
SP - 409
EP - 414
VL - 114
IS - 1
DP - DeepDyve
ER -